Human Immunodeficiency Virus (HIV-1) represents a global threat to public heath and an unprecedented challenge to drug development. Most HIV-1 drug development has focused on viral targets. Unfortunately, given the rapid generation of viral resistance, there have been many failures employing this strategy. It is imperative that new therapeutic modalities be developed. Another approach to take is to develop drugs against host cell gene products that are essential to the productive life cycle of the virus, but not to the host itself. These antiviral targets are potentially broad spectrum against both the wild type and the mutated drug resistant HIV-1 strains. It would be also difficult for the virus to develop resistance against cellular functions required for viral production. Functional Genetics proposes here to use its proprietary technology, Random Homozygous Gene Perturbation (RHGP), to discover novel host cell targets that enable cells to become resistant to HIV-1 infection. This strategy has been used successfully to identify genes whose homozygous perturbation leads to the ability of host cells to resist influenza infection. In this current SBIR Phase I proposal, we plan to use RHGP to randomly inactivate or activate genes in HIV-1 permissive cells, one gene perturbed in each cell, and then screen for cells that acquired resistance to HIV-1 infection as a result of RHGP. We will identify and isolate corresponding host genes in resistant cells. The initial validation of these target genes will be rapidly achieved through a built-in reversible control of the gene perturbation mechanism. Hence, the causal relationship between the perturbed gene and the HIV-1 resistant phenotype can be established as the phenotype reverses with the reversal of the gene perturbation. In Phase II, functional validation and characterization will be performed on these host genes by recreating the gene perturbation in na[unreadable]ve cells to determine roles of their encoding products in the pathogenesis of HIV-1 and to evaluate their potential as therapeutic targets. New drug candidates against HIV-1 infection based on these host targets will then be screened and developed. The major advantages of RHGP technology over other gene inactivation or gene perturbation technologies are that RHGP provides rapid genome wide gene perturbation in a short period of time of a few months. RHGP can cause either gene inactivation through severe knockdown of expression or gene activation through over-expression. There is no requirement of prior knowledge of the gene's identity or function for RHGP to target. RHGP is a reversible gene perturbation method that offers instant validation, through addition of an inducer chemical, of the causal relationship between the observed phenotype, in this case HIV resistance, and the perturbed gene. The repopulation of RHGP integrated cell clones survived from infection-induced killing indicates that the targeted gene products are not essential to cell growth but suitable for HIV-1 drug targets. Most current AIDS drugs target the HIV virus and therefore enable the development of drug resistance through viral mutation. This project seeks to identify human host targets (as opposed to HIV virus targets) that will prevent the HIV virus from using the host's cellular mechanism for its life cycle. Because host mechanisms essential to the virus are blocked, it will be difficult for viral mutations to recreate the essential host mechanism, hence the emergence of drug resistance will be minimized. [unreadable] [unreadable] [unreadable]